Recent technological advances showed that m6A methylation is abundant in human mRNA and long non- coding RNA and can guide a wide range of cellular processes. The HIV genome comprises numerous putative m6A modification sites and our preliminary experimental data show that m6A modifications are present in the HIV genome. The overall goal of the proposed research is to pinpoint the precise sites of m6Amodifications within the HIV genomic RNA and to determine the functional impact of these modifications on HIV biology. In Aim 1 we will apply Site-specific Cleavage And Radioactive-labeling followed by Ligation-assisted Extraction and Thin-layer chromatography (SCARLET), to quantify and precisely locate m6A modifications at a single nucleotide resolution. In Aim 2 we will study the effects of m6A RNA modifications on HIV infectivity and viral production. In addition, we will test the effect of varying m6A in HIV on the immunogenicity of HIV by infecting monocyte derived dendritic cells. The proposed experiments will characterize a novel mode of RNA modification of HIV RNA. The characterization of HIV m6A modifications and its elucidation of its role in the HIV life cycle would not only expand our knowledge of HIV, but also for other viruses that are m6A modified.